Therapeutic Drug Monitoring

Therapeutic drug monitoring (TDM) is an established clinical specialty in which laboratory specialists quantify drug concentrations for the purpose of evaluating therapeutic response. Examples of drugs that are frequently subjected to TDM are antibiotics, antiarrhythmics, antiasthmatics, antidepressants, antiepileptics, and antineoplastics. These drugs possess a narrow range of therapeutic and safe plasma concentration. Therapeutic index (TI) is defined as the ratio between the maximum and minimum plasma concentrations of the drug's therapeutic range. A narrow range is defined as a ratio of 2 to 3. A TI below 2 infers that the dose that yields a subtherapeutic response is close to the dose that produces toxicity. Most drugs have a TI of greater than 2.

The preferred technique for drug analysis is use of an immunoassay analyser (e.g. fluorescence polarization immunoassay, FPIA) because it is relatively quick, can be automated, and requires minimal technician training for execution. However, newer drugs requiring plasma quantification often do not have an immunoassay developed, so chromatography is almost always used at first. Drugs whose metabolites play a role in their efficacy and clinical interpretation also need to be determined by chromatography, which analyses multiple components simultaneously. Immunoassays are often not selective enough to distinguish between parent drug and metabolite, and interferences can adversely affect results for some drugs. Another difficulty facing clinical laboratories is that more accurate quantitation and detection requirements must be satisfied owing to lower dosages being administered. The proliferation of new drugs also increases the potential for concomitant administration. When the issue of cost is considered, in addition to the drawbacks listed above for immunoassays, chromatographic techniques can become quite attractive. Most often for clinical bioanalysis, detection methods involve ultraviolet or fluorescence detection coupled with liquid chromatographic separation.

Antidepressants

Tricyclic and newer antidepressant drugs are one of the most frequently monitored classes of therapeutic drugs in the clinical setting. Drug concentrations are monitored in patients for compliance and to ensure that therapeutic blood levels are reached. Also, patients sometimes take multiple antidepressant drugs, often from different physicians, which can be determined from a LC analysis. Immunoassays frequently cross-react with these drugs (e.g., imipramine, amit-riptyline, desipramine and fluoxetine) and their metabolites. SPE has been used in some immunoassay kits to measure specifically one tricyclic drug. Overall, LC is advantageous because of its ability to monitor simultaneously multiple drugs and to resolve potential interferences from concomitantly administered drugs.

Solid-phase extraction methods for antidepressant drugs abound in the literature. These drugs are basic and can be adsorbed to reversed-phase sorbents such as C18 and C8 by both reversed-phase attraction and secondary interactions via cationic adsorption to silanols on the silica surface. Polar sorbents such as cyanopropyl, in which the cationic adsorption be comes primary, have also been used successfully. Methods for these drugs typically involve a solvent exchange of organic eluent for aqueous/organic mobile phase. Evaporative losses are always a concern with this step, but can be avoided by use of SPE discs, in which elution is accomplished with a small volume of mobile phase solution.

Corticosteroids

The measurement of steroids (prednisone, cortisone, prednisolone, cortisol, corticosterone, methylpredni-solone) in blood is often inaccurate owing to interference from sample matrix and cross-reactivity with chemically similar steroids. For example, antiserum for cortisol is nonspecific and cannot differentiate between cortisol, its metabolites and therapeutically administered steroids. Again, SPE is a preferred technique for drug sample preparation. An efficient method has been reported using C8 sorbent discs, which allows for elution in mobile phase compatible solution for direct injection, eliminating the need for a tedious dry-down and reconstitution step. Steroids are released from proteins by incubation at room temperature with a HCl solution. Neutralization is accomplished by addition of a sodium borate solution. Following centrifugation, the supernatant is loaded onto conditioned C8 discs. A dilute methanol-water solution acts as an efficient wash to remove adsorbed proteins. Elution is performed with acetonitrile, followed by water. The resulting mixture is compatible with mobile phase for direct injection.

Cyclosporin

Cyclosporin, an immunosupressant drug, has many metabolites and is commonly monitored for drug concentrations in the blood of patients who have received an organ transplant. Monoclonal antibody-based immunoassays are in use for this assay, as well as LC methods. Technology for immunoassay detection is constantly being improved, and the tests in use now are reliable. However, LC methods also function quite well, and the issue of antibody versus LC method often rests with cost analysis for a clinic. LC methods rely on SPE using whole blood and, when coupled with automation, can be quite cost effective. An advantage of LC is that it can simultaneously measure several metabolites.

The extraction procedures for cyclosporin are commonly reversed phase. Note that whole blood is preferred to avoid temperature-dependent cyclo-sporin redistribution. Mixing with acetonitrile-water haemolyses blood, and aliquots of the supernatant are loaded onto conditioned extraction columns. Wash steps may involve a weak concentration of acetonitrile in water and elution is achieved with methanol or ethanol, or with an alcohol-water solution.

Antiepileptics

Plasma concentrations of antiepileptic drugs are often monitored during therapy, since a therapeutic range has been well defined. These drugs include phenytoin and carbamazepine and their metabolites, phenobarbital and newer agents such as lamotrigine. Solid-phase extraction is commonly performed using reversed-phase sorbents such as C8 and C18. The wash is performed with water, since the more hy-drophilic drug lamotrigine is removed from the sorbent bed at low organic concentrations in water. Elution is efficiently accomplished with acetonitrile. Again, the use of the disc SPE formats can allow elution in volumes small enough to eliminate the evaporation step; a small elution volume of acetonit-rile is mixed with water and the resulting solution is injected directly onto the liquid chromatograph.

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